Each year approximately 5,000 individuals develop severe enough hepatic failure to require hepatic support. Of these patients, less than 1,000 will undergo an orthotopic liver transplantation which is currently the only available method for the clinical management of severe hepatic failure. For patients who are not selected for transplantation, there is no adequate treatment available. Those suffering from cirrhosis fight the sixth leading cause of death in the United States, and those suffering from acute liver failure face a mortality rate of greater than 80%. There is thus, a critical need for both temporary and more permanent modes of liver support. Non-biological approaches for providing liver support have not succeeded and biological approaches have also been unsuccessful due largely to the inability to keep liver tissue functional in vitro for long periods. The long-term objective of the proposed research is to investigate and establish the liver support systems that could be used to treat acute and chronic liver failure. Our preliminary results have indicated that, by simply sandwiching hepatocytes in between two layers of collagenous matrix, hepatocyte morphology and function are maintained for periods up to 8 weeks. The specific aims of our proposed studies are to: (1) to develop and characterize continuous flow bioreactors using sandwiched hepatocyte layers, (2) to develop optimal hypothermic and cryopreservation protocols for sandwiched hepatocyte layers, and (3) to investigate the use of a larger-scale bioreactor in studies with several small animal liver impairment models. The principles developed in this work will provide a rational basis for the design of an artificial liver device for treatment of liver failure. Additional potential applications of this work include: (1) a stable system for studying hepatocyte growth and differentiation, (2) toxicology testing, (3) hepatocyte cryopreservation, (4) hepatocyte transplantation, and (5) hepatocyte bioreactors for the production of therapeutically valuable proteins.